The promotion of self-association of horse-heart cytochrome c by hexametaphosphate anions

Fur J Blochem 19Y. 561 -568 (1991) ( I-EBS 1991

001 429 5691 004745

The promotion of self-association of horse-heart cytochrome c by hexametaphosphate anions David WHITFORD'. David W. CONCAR', and Robert J. P. WILLIAMS' Departmcnts of Biochcmistry and Inorganic Chemistry Laboratory ', University of Oxford, England

(Rcceived January 23, 1991) - EJB 91 0128

In the presence of the highly charged hexametaphosphate anion, horse heart cytochrome c aggregates to form stable protein complexes. The formation of protein aggregates has been detected by high-resolution 'H-NMR spectroscopy from an increase in the linewidth of resolved ferricytochrome c resonances with hexametaphosphate concentration. Alternatively, analytical ultracentrifugation reveals protein association from the increase in apparent sedimentation coefficients of cytochrome L' in the presence of equimolar hexametaphosphate. Protein aggregation is dependent on the concentration of background electrolyte since in the range 10- 150 mM sodium cacodylate alternative stabilisation of dimeric and trimeric complexes was observed by both NMR and analytical ultracentrifugation. A model is proposed for the mechanism of protein aggregation caused by polyphosphate binding to the surface of cytochrome c.

The crystal structures of eukaryotic cytochromes c provide a wealth of detailed information on the tertiary structure of the protein [ I -41. A distinctive feature of these structures is the asymmetric distribution of positively charged residues. Particularly prominent are lysine residues that surround the haem edge region of the protein. These lysines are thought to be important in electrostatic interactions governing the association of eukaryotic cytochrome L' with both anionic protein and non-physiological redox partners [5 ,6] . In general, these lysine residues show a high level of conservation in proteins from different eukaryotic sources [7].

Evidence for the involvement of charged protein complex formation comes from the ionic strength dependency of the association between cytochrome c and cytochrome bsr cytochrome c peroxidase, or cytochrome oxidase [8 - 101. High ionic strengths lead to a dissociation of these protein complexes, consistent with the interaction of oppositely charged domains, and a decrease in reactivity. Further evidence for the involvement of lysine residues in complex formation with cytochrome L' has come from chemical modification studies. Using a wide variety of reagents that modify lysines, previous workers have shown that only those residues located on the surface close to the haem edge are significantly involved in the interaction of the protein with negatively charged redox partners [ I 1 - 151.

In addition to its role in promoting the association of protein redox partners, the high density of surface lysine residues of cytochrome c presents a large interface for the binding of negatively charged small molecules, particularly transition metal anionic complexes and inorganic phosphates [16, 171. A consequence of the positively charged surhce of cytochrome c is that protein self-association is slight. This is manifest as a slow electron self-exchange rate (< 1000 M- ' s - ', at 300 K) at low ionic strengths (< 100 mM sodium cacodylate) [IS-

Corrcspondmw l o D. Whitford, Dept. of Biochemistry, Univer- sity of Oxford, South Parks Road, Oxford. OX1 3QU, England

~~~~~

201. A significant increase in the rate of electron self-exchange has been observed by either site-specific modification of a lysine close to the haem edge or by the addition of multivalent anions capable of binding to the protein's surface [21]. Although the attachment of a simple anion such as phosphate to the surface of the protein, or the replacement of the amino group of lysine with a carboxylate, facilitates the close ap- proach of two cytochrome c molecules by reducing intermolecular electrostatic repulsion, the self-exchange reactions remain bimolecular processes.

A greater understanding of biological electron transfer will result from a study of unimolecular self-exchange reactions within protein complexes of cytochrome c where the binding, structure and dynamics of reaction partners is previously well characterised. This paper therefore presents a detailed study of the interaction of cytochrome L' with polyphosphate anions, such as hexametaphosphate, which carry a large negative charge at physiological pH and promote protein self-association. Self-association is determined using both nuclear magnetic resonance (NMR) and analytical ultracentrifugation and has been shown to involve electrostatic interactions. The relative stoichiometry of anion binding to cytochrome c, its dependence on solution conditions, and a consideration of the sedimentation coefficients of protein aggregates allows a scheme for polyphosphate-induced aggregation of cytochrome L' to be proposed. It is shown that solution conditions can be optimised to allow protein dimerisation to be the dominant aggregated speices. The identification of a strong polyphosphate binding site on the surface of the protein by para- magnetic difference spectroscopy (described in the following paper) allows the model proposed for protein aggregation to be assigned to particular charged regions of the surface of the protein. A high-affinity domain for hexametaphosphate on the surface of cytochrome c near to the haem edge is identified. The influence of these domains on polyphosphate-catalysed electron self-exchange of cytochrome c is the subject of the

562

third paper and is of particular relevance to current theories of protein electron transfer.

MATERIALS AND METHODS

Protein preparation

Horse heart cytochrome c (Sigma Chemical Co. type VI) was purified to remove polymeric and deamidated forms by cation-exchange chromatography on carboxymethyl-cellulose CM-32 (Whatman Biochemicals Ltd) [22] after initially oxidising the protein with potassium ferricyanide. The eluate was concentrated and exchanged into 20 mM phosphate pH * 7.0 using an Amicon ultrafiltration cell equipped with a YMlO membrane filter (pH * = pH uncorrected for deuterium isotope effect). The protein was stored in a concentrated, oxidised, state at - 20" C until required.

a

I I I

Inorganic reagents 35 30 25 20 15 10 Chernrcal Shift (ppm) Sodium hexametaphosphate (Koch-Light) was used with-

out further purification. Sodium cycodylate solutions in D 2 0 (99.8% Merck Sharp and Dohme) were prepared at the appro- priate pH by the addition of NaOD to cacodylic acid (Sigma

Fig. 1. 600-MHz ' H-NMR spectra of horse heart ferricytochrome c comparing the effect of sodium chloride, sodium hexametaphosphate andpotassium hexacyanocobalt(1ZIj on spectral resolution. All spectra were measured at 300 K with a protein concentration of 2 mM in 20 mM phosphate, pH * 7.0. (a) Spectrum recorded in the presence of 200 mM NaC1. (bj Soectrum recorded in the Dresence of 1 molimol

Chemical Co.).

Sample preparation

Fully reduced cytochrome c samples for NMR were prepared by adding sodium dithionite to the purified protein dissolved in 20 mM cacodylic acid/NaOD pH * 7.0. The reduced protein was further concentrated and exchanged into DzO buffers by repeated ultrafiltration in Centricon microconcentrators (Amicon Ltd) at 4°C. The excess sodium dithionite is rapidly removed by this method and the reduced protein was stored at - 20" C in cacodylate buffer. To prevent any subsequent oxidation of ferrocytochrome c, the samples were kept under a nitrogen atmosphere. When assayed spectrophotometrically these samples showed that over 95% of the total protein remained in a reduced state. Oxidised samples were prepared from the purified protein by ultrafiltration in Centricon microconcentrators to exchange the protein into cacodylate-based buffers.

Protein concentrations were measured spectrophotometrically at 550 nm assuming an absorption coefficient of 29 mM-' cm-' for the fully reduced protein. A sample volume of 400 pl was used in all NMR studies. Sodium hexametaphosphate were added to cytochrome c samples from concentrated stock solutions which were deoxygenated prior to use with reduced protein samples.

N M R methods

'H-NM R experiments were performed using 500-MHz and 600-MHz Bruker-AM spectrometers operating in Fourier-transform mode and equipped with Aspect 3000 com- puters. In order to measure linewidths of hyperfine-shifted resonances, free induction decays were accumulated with spectral widths of 50 kHz (600 MHz) over 16K data points. A delay times of z 1 s was routinely used between the accumu- lation of spectra for the reduced protein. No resolution-en- hancement functions were applied to the free induction decays prior to transformation.

\ ,

hexametaphosphate. (c) Spectrum recorded in'the presence of 1' mol Co(CN)j - /mol

Analytical ultracentrifugation

Analytical ultracentrifugation was performed on a Beckman model E centrifuge fitted with an An-H rotor with a double-sector cell of centrepoint thickness 3.0 mm. The sample volume of approximately 150 p1 containing 1 mM cytochrome c in cacodylic acid/NaOH pH 7.0 was centrifuged at 44000 rpm for approximately 75 min. An equimolar level of hexametaphosphate was added to some samples. Schleiren optic profiles, enhanced through the use of a red filter, were photographed at 4-min intervals over a time period of approximately 40 min. Apparent sedimentation coefficients were calculated from the migration distance and then corrected to a value at 20'C by the following equation:

Values of v and Q at various temperatures were obtained from standard data whilst a value of 0.715 g cm-3 was assumed for the partial specific volume o of cytochrome c [23, 241. The change in viscosity of the solution in the presence of 1 mM protein was neglected and the value for the pure solvent used.

RESULTS

Titrations of cytochrome c with sodium hexametaphosphate

The addition of an equimolar level of sodium hexametaphosphate to horse heart ferricytochrome c results in considerable line broadening of resonances throughout the spectral range. This broadening is revealed most obviously in the loss of spectral resolution of the resolved hyperfine-shifted resonances of ferricytochrome c (Fig. 1). This loss of spectral

563

200 -.

I I

I I I I I I

35 30 25 20 15 10 Chemical Shift cppmt

h ( M e t 80)

B A 2 . F m

1.5

-20 -25 -30 Chemical Shift (ppmt

Fig. 2. 600-MHz N M R spectra showing the effect of successive ad- ditions of hexametaphosphate on the linewidths of hyperfine-shijted resonances of horse heart ferricytochrome c. The titration was carried out by adding hexametaphosphate to the desired concentration of a single sample of 2 mM protein in 20 mM sodium cacodylate, pH * 7.0. Temperature, 300 K. (A) Resonances (a-g) have been assigned to the following resonances: (a) haem C18 methyl; (b) haem C7 methyl; (c) haem C17 propionate r ; (d) Hisl8P; (e) Met808; ( f ) haem C17 propionate r ; (g) haem C12 methyl. (B) (h) Met80~

resolution is not observed in the presence of either high salt concentrations such as 200 mM NaCl or multivalent anions such as potassium hexacyanocobalt(II1). Thus it would appear that sodium hexametaphosphate, unlike the other anions, promotes aggregation of cytochrome c.

To understand the stoichiometry and ultimately the mechanism of hexametaphosphate binding to cytochrome c, the behaviour of the NMR linewidths as a function of hexametaphosphate concentration was measured (Figs 2 and 3). In the oxidised protein several resonances, assigned to haem and haem ligand groups [25, 261, were observed and their linewidths measured throughout a titration. It is clear from Fig. 2 that all the hyperfine-shifted resonances of ferricytochrome c show line-broadening. The effect is not restricted to particular hyperfine-shifted resonances or to individual regions of the protein. The results of Fig. 2 can be more quantitatively expressed as shown in Fig. 3 where it is clearly seen that all the resonances exhibit a maximal value for line- broadening at a hexametaphosphate/cytochrome c (H,) molar ratio of 1.5. At anion/cytochrome c ratios above this level,

X

x

x X - x x x

x e

- c

$ 5 0 . I T . I I

0.5 1.0 1.5 2.0 2.5 3.0

H m Fig. 3. Plots of the effect of hexametaphosphate concentration on the. linewidths of hyperfine-shifted resonances of horse heart ferricytochrome c. The titration was performed on a single sample of 4 mM protein in 20 mM cacodylate(Na0D pH* 7.0 Temperature, 300 K. H,,, is molar ratio of hexametaphosphate/cytochrome c. Resonance a-e as for Fig. 2

the linewidths of all resonances decrease slowly. The results indicate that under the conditions of the NMR experiment (20 mM background electrolyte, 2 mM protein concentration) cytochrome c becomes saturated with hexametaphosphate at low anion concentrations and that an excess of the anion leads to a decrease in oligomerisation. It appears therefore that the affinity of hexametaphosphate for cytochrome c is such that stoichiometric amounts of the anion are sufficient to effect protein aggregation.

Previous workers have suggested that anion binding to cytochrome c is dependent on the protein's oxidation state [27]. 'H-NMR linewidth titrations of cytochrome c in the presence of hexametaphosphate were therefore repeated on samples of the fully reduced protein. Although in its diamagnetic reduced state the protein gives relatively few resolved resonances in its 'H-NMR spectrum, the upfield-shifted resonance of the Met80 emethyl group (6 = -3.25 ppm) was sufficiently well resolved to use as a diagnostic peak. The linewidth of this peak in response to hexametaphosphate (Fig. 4) behaved similarly to that of ferricytochrome c linewidths with maximal broadening occurring in the presence of 1.5 mol/anion/mol. It can be assumed therefore that hexamet- aphosphdte binds similarly to ferro- and ferricytochrome c.

Further characterisation of hexametaphosphate binding and its effect on the self-association of cytochrome c required information about the order of the aggregation resulting from hexametaphosphate binding, the ionic strength dependence of the induced aggregation and the number and location on the protein surface of any strong hexametaphosphate binding sites. The order of aggregation could be estimated from the corresponding increases in linewidths for resolved resonances of ferricytochrome c. Unfortunately the relationship between linewidths and molecular size is complex because the fluctuat- ing magnetic interactions, which cause transverse relaxation, are modulated by both the rate and the anisotropy of molecular reorientation in solution. For a molecule containing an

564

t I

0 I 2 3 Hm

I I I I 0 0.5 1.0 1.5 2.0 2.5

Hm

Fig. 4. Plot of the effect of hexametaphosphate an the lineividths of the Met80 mc~thj~l resonance in f i r r i - and,ferroc:vtochrore c. All titrations were carried out with 2 mM protein in 20 mM cacodylate/NaOD pH* 7.0, Temperature, 300 K . H , is the molar ratio of hexameta- phosphateicytochrome c. (a) Met80 methyl linewidth as a function of H,,, for ferricytochrome c. (b) Met80 methyl linewidth as a function of H , for ferrocytochrome c

electronic magnetic moment, such as ferricytochrome c, the case is further complicated by the need to consider electronic contributions to proton spin - spin relaxation. Thus, although the measured trends in hexametaphosphate-induced line- broadening have a definite relationship to changes in protein mobility, the complexity of this relationship precluded a re- liable and quantitative interpretation in terms of molecular mass of the resultant complexes.

Analytical ult racen t r if ugu t ion q f cy t ochrome c complexes The order of cytochrome L' aggregation was therefore in-

vestigated using analytical ultracentrifugation where the rate of sedimentation of proteins is proportional to their molecular mass. Fig. 5 shows the effect of hexametaphosphate on the sedimentation velocity of ferricytochrome c at both low (20 mM) and high (140 mM) levels of sodium cacodylate. The apparent sedimentation coefficient (s) of approximately 1.6 S determined in the absence of hexametaphosphate compares favourable with that obtained previously for monomeric cytochrome c. From the profiles shown in Fig. 5 the dependence of the sedimentation coefficient on hexametaphosphate concentration is nearly linear up to the addition of 1.0 mol hexametaphosphate/moI as seen in NMR experiments. Unlike the NMR experiments above, a zero order dependency is

a

0.5 1.0 1.5 2.0 2.5

Hrn Fig. 5 . The effect of/ie.~ametaphosphate on the apparent sedimcwtution coqfjcient offerricytochrome c measured by analj~tical ultracentrifugation. Each point represents the sedimentation coefficient calculated from a minimum of 12 Schlieren photographs at 4-min intervals. The Centrifugation was carried out at a protein concentration of 1 mM in cacodylate buffer pH 7.0. H , denotes the amount of hexametaphosphate added (mol/mol). (a) 20 mM cacodylate/NaOD pH * 7.0; (b) 120 mM cacodylate/NaOD pH * 7.0

observed between 1 .O - 2.0 mol/mol. This trend occurred ir- respective of the ionic strength of the solution. It should be noted that, although similar titration profiles were obtained, the magnitude of the sedimentation coefficient estimated in a background electrolyte of 20 mM sodium cacodylate (saPp = 5.0 S) was far greater than that predicted for a dimeric aggregate of two 12-kDa subunits. At higher levels of cacodylate (140 mM) a sedimentation coefficient of 2.7 S was estimated. This lower value appears to indicate that hexametaphosphate- induced aggregation is limited at higher cacodylate concentrations and that the overall aggregation process is influenced by electrolyte levels and depends on electrostatic interactions with the protein - anion complex.

The ionic strength dependence of oligomerisation To confirm the presence of discrete oligomers of cyto-

chrome c at different ionic strengths, analytical centrifugation was performed over a wide range of background electrolyte (cacodylate) concentrations. Fig. 6 shows the effect of increasing ionic strength on the apparent sedimentation coefficient. Although some variation in calculated sedimentation coefficients occurs, three distinct plateaus can be seen at cacodylate concentrations between 20-40 mM, 60-90 mM and 100- 140 mM. Within these concentration ranges, the sedimentation coefficients do not vary significantly with electrolyte concentration. This result suggests a stabilization of the aggregated species of cytochrome c giving apparent sedimentation coefficients of 5.4 S, 3.3 S and 2.7 S respectively. At very high levels of cacodylate buffers ( I = 0.3 M) the sedimentation coefficient approximated to that observed in the absence of hexametaphosphate, suggesting that excess background electrolyte can suppress protein aggregation.

To verify the dependence of oligomerisation of cytochrome c on the concentration of background electrolyte, the line-

565

5 t + + + + * + + + +

+ + + I I I

0 40 80 120 [Na'Cac'] (mM)

Fig. 6. The effict of cacodylate concentration on the sedimentation coefficient of ,ferricvtochrome c in the presence of equimolar hexa- metaphospliute measured using analytical ultracentrijiugation. Appa- rent sedimentation coefficients were recorded for 12 samples each containing 1 mM protein at different concentrations of sodium cacodylate (pH 7.0) (Na'Cac-). Each sedimentation coefficient (values in S) was calculated from at least 12 Schlieren profiles. The dotted line reflects the sedimentation coefficient of monomeric cytochrome c

4 0 1 , , . , . , . , 1 1 8 0 1 , , , , , , , , J 0 40 80 120 160 0 40 80 120 I60

[Na'Cac-] (mM) [Na'Cac-] (mM)

Fig. 7. The ej'ect of cacodylate concentration on the linewidths of haem C18 and C7 methyls and Met80 &-methyl offerricytochrome c measured in the presence of equimolar hexametaphosphate. The titration was carried out on a single sample of 1 mM protein. The ionic strength was increascd by raising the concentration of background electrolyte sodium cacodylate (pH* 7.0). The linewidths were recorded at 600 MHz and 300 K

widths of resonances of ferricytochrome c were determined in the presence of equimolar ratios of hexametaphosphate at a range of ionic strengths. An anion/protein molar ratio of 1 .O was used in the experiments since analytical ultracentrifugation indicated that a single mole of hexametaphosphate is involved in the primary aggregation process of a mole of protein. Raising the ionic strength (5- 15 mM cacodylate) leads to an initial sharp decrease in the linewidths of the haem C18 methyl (formerly designated haem methyl 8), haem C7 methyl (formerly designated haem methyl 3) and Met80 methyl resonances of ferricytochrome c (Fig. 7). This was arrested at intermediate ionic strengths (20-40 mM, 60- 90 mM, 100 - 140 mM) to give a stepwise motional narrowing of the linewidths. For the haem C7 methyl resonance, the

80

70

- N X - 60 a

50

LO

30

r/

I

1 2 3 Hm

Fig. 8. The effect of hexametaphosphate concentration on the lineit.idth.s qf haem C18 and C7 methyls of ferricytochrome c in the presenci~ oJ high concentrations of sodium cacodylate. Titrations were carried out on a 1 mM sample of protein in 120 mM cacodylate/NaOD pH * 7.0. Spectra were recorded at 600 MHz and at 300 K. (a) CIS methyl; (b) C7 methyl. H,,, is the molar ratio of hexametaphosphate/cytochrome ('

curve decreases rapidly before giving two distinct steps at linewidths of 65 and 53 Hz whereas the Met80 gives rise to steps at 240 and 280 Hz. At high concentrations of electrolyte the linewidths of all three resonances tended towards values characteristic of the monomeric protein.

Taken together, the NMR data and the analytical ultracentrifugation results provide evidence for three distinct, self- associated, states of cytochrome c present at a uniform equimolar concentration of hexametaphosphate but at different cacodylate concentrations.

The dependence of oligomerisation on solution conditions

Hexametaphosphate binding to the surface of cytochrome c clearly promotes protein aggregation in a process which is controlled by the levels of background electrolyte. To clarify the mechanism of hexametaphosphate binding, the titrations of Fig. 3 were repeated in the presence of 120 mM sodium cacodylate where the lowest order aggregate, or dimeric complex, is the dominant species. NMR linewidths for the haem C18 methyl and haem C7 methyl resonances, measured as a function of hexametaphosphate concentration, show a linear dependence up to the addition of 1 .O mol hexametaphosphate/ mol (Fig. 8). The subsequent linewidth increases above 1 mol/ mol are more gradual than those obtained at lower cacodylate concentrations and suggests that a single strong hexametaphosphate binding site is responsible for protein dimerisation. At higher cacodylate concentrations, a sup- pression of secondary binding occurs with a decrease in the order of aggregation.

From the results of the ionic strength dependency of NMR linewidths and sedimentation coefficients it is observed that, for a fixed protein concentration, the level of background electrolyte may be used to modulate protein self-association in the presence of hexametaphosphate. This can lead, for example, to the selective formation of cytochrome c dimers.

566

I I I 1 2 3 4

[FemCc] (mM I

- 2 4 6 8

[FemCc] (mM)

Fig. 9. The effect ofprotein concentration on the linewidths of haem C18 and C7 methyls of ferricytochrome c measured in the presence of equimolar hexametaphosphate. All spectra were recorded at 600 MHz, temperature 300 K. (a) The variation of linewidth of haem C1S ( W ) and C7 (V) methyls with protein (ferricytochrome c) Concentration, [FeIIICc], at a constant cacodylate concentration of 120 mM. (b) The variation of linewidth of haem CIS (V) and C7 (m) methyls with protein concentration at a constant electrolyte/protein ratio of 60

It is important, however, to determine whether the critical factor in this cacodylate dependency was the relative electrolyte/protein concentration or the formal ionic strength of the solution. NMR linewidths were therefore measured at a range of different protein concentration (0.25 - 8 mM ferricytochrome c) in the presence of 1 .O mol hexametaphosphate/mol and with either a constant sodium cacodylate concentration of 120 mM or an electrolyte/protein ratio of 60. Maintaining the sodium cacodylate concentration constant resulted in a decrease in the resonance linewidths as the protein concentration was lowered between 4 - 0.25 mM (Fig. 9). In contrast, when the electrolyte/protein ratio is kept constant, the linewidths increase as the protein concentration is lowered below 2 mM although their initial sensitivity to a lowering of the protein concentration is less marked. This result indicates that it is the relative concentration of the electrolyte which is important in controlling the self-association process and not its contribution to the formal ionic strength. Furthermore, the observation of tight binding of a single hexametaphosphate molecule to a molecule of cytochrome c at both high and low formal ionic strengths is significant in terms of the stepwise aggregation mechanism.

DISCUSSION The similarity of the chemical shifts of the hyperfine reso-

nances in the presence and absence of hexametaphosphate confirms that anion binding is not accompanied by any major perturbations in the tertiary structure of cytochrome c. Protein aggregation, promoted by hexametaphosphate anions, involves the native folded state for cytochrome c and is indepen- dent of the redox state of the protein. This is in general agree- ment with NMR and crystallographic studies suggesting that,

for the aromatic and the main-chain regions of the protein, conformational changes between the reduced and oxidised forms are slight [2,28]. The origin and mechanism of differential anion binding to reduced and oxidised cytochrome c is unclear and, if it is present under the current experimental conditions, does not modulate the overall process of protein aggregation.

The most logical mechanism for hexametaphosphate-induced oligomerisation involves the binding of the anion to the surface of cytochrome c, thereby reducing intermolecular electrostatic repulsion and facilitating protein self-association. Both NMR and analytical ultracentrifugation indicate that protein association is elicited by stoichiometric amounts of hexametaphosphate and that the primary mode of association involves a single site for the anion on the surface of cytochrome c. However, at low ionic strengths the behaviour of NMR linewidths, with increasing hexametaphosphate concentration, is complicated by the apparent increase in aggregation state above 1 mol anion/mol and the interdependence of hexametaphosphate affinity for the protein on the ionic strength of the solution. The increases in linewidth above 1 mol anion/mol can be attributed to secondary binding of hexametaphosphate on the surface of cytochrome c. These secondary sites of interaction between hexametaphosphate and cytochrome c are suppressed at higher cacodylate concentrations as seen from the decreased extent of oligomerisation observed by both NMR and analytical ultracentrifugation.

An understanding of the mechanism of hexametaphosphate binding requires a consideration of the role of background electrolyte in modulating protein oligomerisation. So- dium cacodylate was used as a background electrolyte/buffer because the cacodylate anion is known to bind weakly to the surface of cytochrome c compared with, for example, chloride. The relative effects of the two anions on the self-exchange rate of cytochrome c further emphasise this point. Direct competition between cacodylate and hexametaphosphate anions is limited in view of the observation of strong (1 : 1) hexametaphosphate-cytochrome binding at both high (120 mM) and low (20 mM) electrolyte concentrations (Figs 6 and 7). It is assumed, therefore, that the main effect of the electrolyte is to limit, by shielding residual exposed charges, the overall process of oligomerisation. Further support for this mechanism is seen in the titrations of the haem C18 methyl resonance of cytochrome c at 120 mM cacodylate where the increases in linewidth above 1 mol hexametaphosphate/mol were decreased in magnitude compared with similar titrations at lower ionic strength (Fig. 8). Further definition of the relationship between ionic strength and hexametaphosphate- induced oligomerisation was shown from thc relative effects of varying the protein concentration (in the presence of equimolar hexametaphosphate) at a constant electrolyte/protein ratio or at a constant electrolyte concentration. The decrease in linewidth of haem C18 and C7 methyls of ferricytochrome c when the electrolyte concentration was kept constant at 120 mM is consistent with the decrease in oligomerisation that occurs as a result of the increase in effective charge shielding. Conversely, the proportional decrease in the concentration of hexametaphosphate-bound ferricytochrome c and electrolyte results in decreased charge shielding with a concomitant increase in the order of oligomerisation. It may be further stated that the effective strength of charge shielding depends on both the electrolyte and protein concentration. The results may be summarised by a two-step model in which hexametaphosphate binding at the high-affinity site is not inhibited by cacodylate but in which electrostatic attraction

567

between hexametaphosphate-bound cytochromes c is modulated by the concentration of background electrolyte.

I f it assumed that weak secondary binding of hexametaphosphate to the surface of cytochrome c is modulated by the background electrolyte concentration and is responsible for the increase in linewidths of resonances observed by NMR above 1 mol hexametaphosphate/mol then, from Fig. 5, it is clear that the sedimentation velocity measured by analytical ultracentrifugation fails to detect such changes. NMR linewidths and sedimentation velocities are both related to protein mobility. For NMR linewidths, the dipolar proton - proton interactions largely responsible for transverse relaxation are modulated by the rotational properties of the protein in solution whereas the sedimentation coefficient is determined predominantly by its translational motion. Although the rates of rotation and translation are dependent on molecular dimensions, it is likely that certain types of interaction exist which are capable of influencing rotational motion as measured by NMR linewidths but not by analytical ultracentrifugation. Indeed recent studies of the interaction between cytochromes h5 and c have emphasised the sensitivity of NMR methods to molecular mobility from differential changes in resonance linewidth within a 1 : I complex [291.

The occurrence of stepwise rather than monotonic decreases in linewidths and sedimentation coefficients within limited ionic strength ranges indicated the formation of at least three distinct oligomeric states of cytochrome c. The sedimentation coefficient of these oligomeric states were in order of increasing molecular size 2.7 S, 3.3 S and 5.4 S. The determination of molecular masses from the sedimentation coefficients of aggregating species requires a consideration of the shape as well as the molecular size of the protein. Theoreti- cal models of the effect of shape on the frictional coefficient have proposed that, for a fixed protein mass of uniform density, deviations from spherical symmetry lead to a reduction in the sedimentation rate [30,31]. The most obvious structural model for a dimeric aggregate of cytochrome c would envisage a complex whose length is twice that of the monomeric complex. Using this simple approximation, most models predict a sedimentation coefficient 1.4- 1.6 times (as opposed to 2 times) greater than the monomeric protein. Thus the sedimentation coefficients of 2.7 S and 3.3 S observed during hexametaphosphate-induced oligomerisation could be equated with dimeric and trimeric protein complexes. Further support for this notion comes from the similarity of values estimated for the sedimentation coefficients associated with the steps in the ionic strength dependency to those obtained previously for covalently bound tetrameric, trimeric and dimeric forms of cytochrome c (231.

A knowledge of the stoichiometry of the hexametaphosphate - cytochrome c complexes at various ionic strengths from analytical ultracentrifugation permits a more accurate interpretation of molecular species responsible for steps attributed to motional narrowing in Fig. 7. For the linewidths of the haem C7 methyl resonance the steps corresponding to trinier and dimer formation have values of 65 Hz and 53 Hz respectively, whereas in the monomeric protein the corresponding linewidth for this resonance is 38 Hz. On the basis of the linewidths of ferrocytochrome c and previous studies of the field dependence of the linewidth of the haem C 7 methyl (321, it is resonable to assume that the diamagnetic contribution to the haem C7 linewidth is z 12 Hz. Thus in the slow tumbling region, where diamagnetic linewidths are approximately proportional to molecular size, the increases in the linewidth of the haem C18 methyl resonance fall within the

expected range for dimeric and trimeric aggregates of cytochrome c.

In conclusion, a mechanism for cytochrome c aggregation in the presence of hexametaphosphate would envisage the binding of hexametaphosphate to the surface of cytochrome c as a result of favourable electrostatic interactions provided by lysine residues around the haem. These hexametaphosphate -cytochrome-c molecules form protein complexes as a result of the formation of surfaces that are conducive to self- association. In view of the observed stoichiometry of hexametaphosphate-induced cytochrome c aggregation, it is un- likely that a single anion can bridge two protein molecules. Instead, protein dimerisation can be viewed as a result of favourable interactions between charged and uncharged regions of two cytochrome c molecules each with a single molecule of hexametaphosphate bound to the surface.

The authors thank Jane Heritage for help with the analytical ultracentrifugation experiments, the Science and Engineering Re- search Council (SERC) and Nuffield Foundations for financial support, and the Oxford Centre for Molecular Sciences for NMR and computational facilities.

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